Oncogenic BRAF induces whole-genome doubling through suppression of cytokinesis

Melanomas and other solid tumors commonly have increased ploidy, with near-tetraploid karyotypes being most frequently observed. Such karyotypes have been shown to arise through whole-genome doubling events that occur during early stages of tumor progression. The generation of tetraploid cells via whole-genome doubling is proposed to allow nascent tumor cells the ability to sample various pro-tumorigenic genomic configurations while avoiding the negative consequences that chromosomal gains or losses have in diploid cells. Whereas a high prevalence of whole-genome doubling events has been established, the means by which whole-genome doubling arises is unclear. Here, we find that BRAFV600E, the most common mutation in melanomas, can induce whole-genome doubling via cytokinesis failure in vitro and in a zebrafish melanoma model. Mechanistically, BRAFV600E causes decreased activation and localization of RhoA, a critical cytokinesis regulator. BRAFV600E activity during G1/S phases of the cell cycle is required to suppress cytokinesis. During G1/S, BRAFV600E activity causes inappropriate centriole amplification, which is linked in part to inhibition of RhoA and suppression of cytokinesis. Together these data suggest that common abnormalities of melanomas linked to tumorigenesis – amplified centrosomes and whole-genome doubling events – can be induced by oncogenic BRAF and other mutations that increase RAS/MAPK pathway activity.

I ncreased ploidy is a common feature of solid tumors. The most frequently observed increased karyotypes approach tetraploidy, which led to the hypothesis that such 'near-tetraploid' tumors had undergone a whole-genome doubling (WGD) event during tumor progression and subsequently experienced a small net loss of chromosomes 1,2 . Recent bioinformatic analyses support this hypothesis, showing that WGD events are prevalent in a diverse set of solid tumors, and nearly 37% of all solid tumors measured, including 40% of melanomas, experienced at least one WGD event in their progression 3,4 . Based on these analyses, WGD frequently occurs early in tumor formation, and the presence of tetraploid cells in some pre-cancerous lesions, such as Barrett's esophagus and lesions of the cervix and kidney, suggests that WGD may even precede frank tumor formation in some tissues [5][6][7][8] Tetraploidy was also observed in hyperplastic lesions of the pancreas 9 , in localized prostate cancer [10][11][12] and some colon adenomas 13,14 , and for certain malignancies, such as oral tumors 15 , tetraploidy is a strong predictor of malignant transformation. Additionally, in established cancers from many tissue types WGD is a predictor of poor clinical outcome 16 .
Tetraploidy has been experimentally linked to tumorigenesis. Viral-induced cell fusion has been shown to enhance the transformation and tumor-forming capabilities of different cell types [17][18][19][20] . Additionally, in mouse mammary epithelial cells that were made tetraploid through treatment with the actin filament poison dihydrocytochalasin B, tetraploid cells were able to form tumors in mice whereas their isogenic diploid counterparts were not 21 . In support of a role for WGD in tumorigenesis, deep sequencing of tumor samples has shown WGD to be an early event in non-small cell lung cancer, medulloblastoma and other tumor types 1,2,22,23 . There are different and mutually inclusive ways in which tetraploidy could contribute to tumorigenesis. First, tetraploidy can enable cells to become tolerant to the negative consequences of chromosome gains, losses, gene deletions, and inactivating mutations [24][25][26][27][28][29][30] . Hence, tetraploidy is likely to allow tumor cells to withstand a higher incidence of mutations, thereby increasing the probability of adaptive changes. Second, tetraploid cells have an increased rate of chromosome missegregation [31][32][33] , thus increasing the possibility that a developing tumorigenic clone will accumulate and tolerate the mutations needed for its progression to a malignant state 34 . Thirdly, proliferating tetraploid cells are genetically unstable and can facilitate tumor progression by giving rise to aneuploidy, a known hallmark of cancer 35 .
Melanomas are a tumor type in which WGD is prevalent 4 . Although molecular genetic analyses have provided great insights into the genes that are involved in melanoma, very little is known about the process by which melanocytes with these lesions become tumorigenic, and whether any mutations underlie WGD in tumors is unclear. We examined melanocytes in zebrafish strains that are predisposed to melanoma and discovered an abundance of binucleate, tetraploid melanocytes. Tetraploidy was caused by expression of BRAF V600E , which increases RAS/MAPK-pathway activity and is commonly found in human melanomas. Using an in vitro model combined with live imaging, flow cytometry and immunofluorescence approaches, we found that BRAF V600E generated tetraploid cells via cytokinesis failure and reduced activity of the small GTPase RhoA, which is critical for cytokinesis 36 . We also show that BRAF V600E activity causes inappropriate centrosomal amplification, which is linked in part to the inhibition of RhoA and suppression of cytokinesis. Additionally, we show that zebrafish melanomas have a tetraploid karyotype and tumor-initiating cells in the zebrafish are tetraploid. These data collectively suggest that BRAF V600E -induced WGD occurs and has a role in tumor formation.

Results
BRAF V600E causes melanocytes in zebrafish to be tetraploid and binucleate. In this and previous studies, we used a zebrafish model of melanoma which combined melanocyte-lineage expression of human BRAF V600E with an inactivating mutation in the endogenous zebrafish p53 gene [37][38][39] . Animals of this genotype, Tg(mitfa:BRAF V600E ); p53(lf), develop melanomas that have histopathological and molecular features similar to those of human melanomas. To determine if tumors arising in this model exhibited ploidies consistent with having undergone a WGD event, we harvested tumors from Tg(mitfa:BRAF V600E ); p53(lf) animals and quantified DNA content. The ploidy of these zebrafish melanomas was predominantly 4N and higher (Fig. 1A), indicating that WGD is likely a feature of this model. The analyzed tumors displayed a small fraction of 2N cells, which we speculate were admixed stromal cells.
To investigate when the WGD event could occur, we began by examining melanocytes from Tg(mitfa:BRAF V600E ); p53(lf) animals. We reasoned that closer analyses of epidermal melanocytes in the Tg(mitfa:BRAF V600E ) strains might reveal the basis of the observed tetraploidy and provide insight into early cellular events that occur in melanoma tumorigenesis. To this end we developed assays to quantify and determine cell biological characteristics of these melanocytes. To quantify dorsal epidermal melanocytes, we treated fish with epinephrine then plucked and fixed scales to which these melanocytes are attached. As the number of melanocytes per scale depends on the size of the scale, we obtained a normalized melanocyte density measurement. Melanomas arise from dorsal regions of these zebrafish, and we found that the scaleassociated epidermal melanocytes in these dorsal regions were larger in size and fewer in number than those of wild-type zebrafish (Fig. 1B, C, Supplementary Fig. 1A). This was due to BRAF V600E expression, as Tg(mitfa:BRAF V600E ) melanocytes were also larger and fewer in number, whereas p53(lf) melanocytes were similar to those of wild-type zebrafish. Previously, injection of BRAF V600E had been shown to cause nevus-like proliferations of melanocytes in zebrafish 38 , and we also showed nevus-like melanocyte proliferations can arise in the Tg(mitfa:BRAF V600E ); p53(lf) strain 37 . However, our current characterization of melanocytes in strains stably expressing the Tg(mitfa:BRAF V600E ) transgene indicates that, aside from a few melanocytes that clonally proliferate, BRAF V600E expression primarily results in a reduced number and increased size of melanocytes. Cell size increases can be caused by increased ploidy, which could be reflected in a larger nuclear size 40 . To determine if BRAF V600E expression caused nuclear enlargement, we stained for the melanocyte nuclear protein Mitfa. Most nuclei in large Tg(mitfa:BRAF V600E ) melanocytes were similar in size to those of wild-type melanocytes; however, melanocytes in Tg(mitfa:BRAF V600E ) contained two nuclei (Fig. 1D, E). Melanocytes from Tg(mitfa:BRAF V600E ); p53(lf) animals were similarly binucleate, whereas p53(lf) melanocytes had one nucleus (Fig. 1E, Supplementary Fig. 1B), indicating that the binuclearity is associated with BRAF V600E expression. Whereas binucleate dorsal epidermal melanocytes are rare in wild-type animals, binucleate stripe-associated dermal melanocytes are more commonly observed 41,42 , suggesting that mechanisms that coordinate nuclear and cellular divisions may be particularly prone to regulation in zebrafish melanocytes.
To determine if the binuclearity we observed was uniquely associated with BRAF V600E or was caused by Ras/BRAF pathway overactivity in general, we stained scale-associated melanocytes expressing a common oncogenic variant of NRAS, mutations in which are present in about 28% of human melanomas 43 . Melanocytes from animals expressing an NRAS Q61L oncogene that is commonly found in human melanomas were also binucleate ( Fig. 1E, Supplementary Fig. 1C) 44 , and binucleation has also been observed in zebrafish melanocytes that express an oncogenic variant of HRAS 45 . Together these data indicate that binucleate cells arise from overactivation of RAS/MAPK signaling.
To examine ploidy of Tg(mitfa:BRAF V600E ) melanocytes, flow cytometry and DNA densitometry were performed. Zebrafish melanocytes retain melanin pigment, so an mitfa:EGFP transgene and albino mutation were introduced so that melanocytes could be reliably identified by GFP-positivity and characterized without melanin spectral interference. Flow cytometry showed that Tg(mitfa:BRAF V600E ) melanocytes were predominantly tetraploid, with small fractions of diploid and octoploid cells observed (Fig. 1F). Flow cytometry also confirmed the binucleate nature of these cells (Supplementary Fig. 1D (Fig. 1G). Therefore, expression of BRAF V600E causes melanocytes in zebrafish to become tetraploid as a result of having two nuclei, each with a 2N DNA content.
BRAF V600E binucleate, tetraploid cells arise via failure of cytokinesis. To determine how BRAF V600E generates binucleate, tetraploid cells and to recapitulate the phenotype we observed in our zebrafish model, we developed an in vitro system suitable for mechanistic analyses. This system is based on a single-cell clone we created in which BRAF V600E was inducibly expressed in RPE-1 FUCCI cells at a level similar to that of endogenous BRAF ( Fig. 2A, B). RPE-1 cells were chosen for this analysis because they are non-transformed, hTERT-immortalized, and have a stable, diploid karyotype. We combined DNA content analysis with the fluorescent ubiquitin-based cell cycle indicator (FUCCI) reporter system to enable abnormal tetraploid cells in the G1 phase of the cell cycle (Cdt-mCherry-positive) to be distinguished from normal tetraploid cells in the G2 or M phases of the cell cycle (Geminin-GFP-positive) 46,47 . In these cells, synchronization was performed by serum starvation, then after serum addition and progression through mitosis BRAF V600E was induced using doxycycline, and cells were synchronized again with a thymidine block. Following release from thymidine synchronization and progression through mitosis, G1 tetraploids were measured as Cdt-mCherry-positive cells with a 4N DNA content (Fig. 2C, Supplementary Fig. 2A). Expression of BRAF V600E caused a nearly three-fold increase in the percentage of G1 tetraploid cells (Fig. 2D). These cells were CyclinD1-positive, confirming that they were in G1 and not G2 cells that had dysregulated Cdt-mCherry expression ( Supplementary Fig. 2B). Expression of wildtype or kinase-dead BRAF showed no similar increase in G1 tetraploids (Fig. 2D). Using a single-cell clone in which BRAF V600E was inducibly expressed in RPE-1 H2B-GFP cells, live-cell imaging was used to investigate whether the G1 tetraploids were binucleate and, if so, how they arose. Indeed, after BRAF V600E induction and release from synchronization, binucleate cells were observed in the following G1 phase of the cell cycle (Fig. 2E, F). These cells arose through failure of cytokinesis characterized by the formation then regression of the cytokinetic cleavage furrow. Modest increases in other mitotic defects, including lagging chromosomes, chromosome bridges and micronuclei, were observed although none of these increases was statistically significant (Supplementary Fig. 2C-E). Mitotic duration was not affected, even in cells that had undergone cytokinesis failure ( Supplementary Fig. 2F). Together these data indicate that oncogenic BRAF V600E causes WGD and the formation of binucleate, tetraploid cells by impairment of cytokinesis rather than cell fusion or other means. BRAF V600E causes cytokinesis failure by reducing the localization and function of RhoA. To understand how BRAF V600E inhibits cytokinesis, we investigated the localization and function of proteins that are involved in mitotic exit and the cytokinetic process. Polo-like kinase I (PLK1) helps to initiate cytokinesis 48 , and it has been shown to interact with CRAF in G2/M 49 . We tested whether cytokinesis failure was dependent on PLK1 and found that an inhibitor of PLK1 did not affect BRAFV600E tetraploid formation ( Supplementary Fig. 3A). MPS1/TTK1, a kinase which is activated by BRAFV600E and promotes activation of the mitotic spindle checkpoint 50 , was not activated in response to BRAFV600E expression in RPE-1 cells and thus not likely involved in BRAFV600E-driven cytokinetic failure (Supplementary Fig. 3B). Other proteins that have been extensively characterized as being critical during cytokinesis, mainly in contractile ring and cleavage furrow formation, are RhoA, a member of the RhoGTPase family and its scaffold protein Anillin 36,51 . Following anaphase Anillin is required to maintain the assembly of cytokinetic furrow components at the equatorial cell cortex. In cells expressing BRAF V600E , Anillin staining was greatly reduced (Fig. 3A, B). Anillin localization is regulated by RhoA 52 , which activates and coordinates several downstream events in the cytokinetic process. RhoA is spatiotemporally activated and accumulates at the equatorial cell cortex in anaphase and during cytokinesis. This accumulation is both necessary and sufficient for cytokinesis to proceed 53 . Similar to Anillin, RhoA localization to the cell equator was reduced in BRAF V600E -expressing cells (Fig. 3C, D). RhoA reduction in BRAF V600E -expressing cells was dependent on increased MAPK signaling because treatment with the MEK inhibitor trametinib or ERK inhibitor SCH772984 restored RhoA localization (Fig. 3C, D). Since RhoA localization is promoted by its activation at the equatorial cell cortex 54 , we quantified the levels of active, GTP-bound RhoA in BRAF V600E -expressing cells. Levels of GTP-bound RhoA were reduced as a consequence of BRAF V600E expression (Fig. 3E, F). BRAF V600E -dependent reduction in GTP-bound RhoA has also been observed in immortalized melanocyte Mel-ST cells, indicating relevance to melanocyte biology 55 . If BRAF V600E acts to reduce function of RhoA, then an increase in RhoA function would be predicted to suppress the effect of BRAF V600E on the formation of binucleate, tetraploid cells. This occurred, as treatment of BRAF V600E -expressing cells with RhoA activators LPA or S1P reduced the formation of tetraploid cells (Fig. 3G). Furthermore, expression of the RHOA Q61L activated variant suppressed BRAF V600E -induced tetraploidy (Fig. 3H). These data indicate that BRAF V600E reduces the activity of RhoA and its downstream effector Anillin, which underlies the failure of cytokinesis and the formation of binucleate, tetraploid cells. causes melanocytes in zebrafish to be tetraploid. A DNA content of normal and tumor tissue from Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); p53(lf); alb(lf) zebrafish. ZF1, ZF2, and ZF3 are three different animals. B Scales from wild-type, Tg(mitfa:BRAF V600E );p53(lf), Tg(mitfa:BRAF V600E ) and p53(lf) strains. Melanin pigment is dispersed throughout the cytoplasm of zebrafish melanocytes, revealing markedly different cell sizes. Scale bar = 250 μm, insets are at same scale as one another. C Quantification of melanocyte densities per scale of wild-type, Tg(mitfa:BRAF V600E );p53(lf), Tg(mitfa:BRAF V600E ) and p53(lf) strains. N = 5 for three animals of each genotype; the mean density of each animal is plotted. One-way ANOVA with Tukey's multiple comparisons test. Error bars represent mean ± SEM. D Images from brightfield (left), anti-Mitfa (middle) and DAPI (right) staining of a single wild-type (top) or Tg(mitfa:BRAF V600E ) (bottom) epidermal melanocyte. Only the melanocyte nuclei stain positively for Mitfa. White arrowheads indicate nuclei within a single melanocyte. Scale bar = 5 μm. Representative cells quantified in E are shown. E Mean percent binucleate cells as determined by anti-Mitfa staining of pigmented melanocytes. N = 3 experiments examining in total wild type = 636, Tg(mitfa:BRAF V600E );p53(lf) = 458, Tg(mitfa:BRAF V600E ) = 454, p53(lf) = 538, and Tg(mitfa:NRAS Q61K ) = 122 melanocytes. One-way ANOVA with Tukey's multiple comparisons test. Error bars represent mean ± SEM. F Flow cytometry and DNA content analysis of control Tg(mitfa:EGFP); alb(lf) and Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); alb(lf) melanocytes with brightfield, EGFP and DAPI images of single melanocytes. G DNA content analysis of wild-type and Tg(mitfa:BRAF V600E ) melanocyte nuclei by confocal densitometry. N = 25 nuclei for wild type and N = 35 for Tg(mitfa:BRAF V600E ). Bars represent median. BRAF V600E and MAPK pathway activity is required during late G1 and early S phases for generating tetraploids. BRAF V600E acts in G1/S to promote cell cycle progression, yet some reports have suggested that MAPK activity is important during mitosis [56][57][58] . To determine when BRAF V600E and MAPK signaling is required to generate tetraploids, we treated BRAF V600Eexpressing cells with the BRAF V600E inhibitor vemurafenib, MEK inhibitor trametinib and ERK inhibitor SCH772984 at various points in the cell cycle and measured whether the inhibitors suppressed tetraploid formation (Fig. 4A). The ability of inhibitors to reduce downstream MAPK activity was confirmed by western blot of phosphorylated ERK ( Supplementary Fig. 4A, B).
Additionally, we also confirmed that treatment with inhibitors at the concentrations used did not cause cell cycle arrest and did not substantially impact growth kinetics, cell cycle progression or cell viability ( Supplementary Fig. 4C-E). Treatment with inhibitors throughout the cell cycle suppressed formation of BRAF V600Einduced tetraploids (Fig. 4A). By contrast, treatment during the S/ G2/M phases had little effect on tetraploid formation. Treatment during G1 and specifically during late G1 and early S suppressed formation of tetraploids. This suppression occurred with each of the three inhibitors tested as well as the 'paradox-breaking' BRAF V600E inhibitors PLX7904 and PLX8394 ( Supplementary  Fig. 4F), which inhibit BRAF V600E while not simultaneously  signaling activity during G1/S could lead to downregulation of RhoA and failed cytokinesis, we considered regulators of cytokinesis that are active during G1 or S phases and whose dysregulation could impair cytokinesis. The small GTPase Rac1 is a negative regulator of cytokinesis that inhibits function of the contractile ring 53 . Rac1 is active throughout the cell cycle except during a small window in mitosis, with its nadir of activity during  63 , and overactivation of Rac1 impairs RhoA localization to the equatorial cell cortex 64 and causes cytokinesis failure leading to formation of binucleate cells 65 . To investigate whether BRAF V600E affects Rac1 activity, we performed ELISA assays to detect active, GTP-bound Rac1 at various points following BRAF V600E expression and release from synchronization. As compared to control cells, GTP-bound Rac1 levels in BRAF V600Eexpressing cells were higher upon release from synchronization and thereafter (Fig. 4B). To determine if higher Rac1 activity contributes to the BRAF V600E -induced formation of binucleate cells, we treated cells with the Rac1 inhibitors NSC2366 and EHT1864 and measured G1 tetraploid formation in BRAF V600Eexpressing RPE-1 FUCCI cells. Treatment with either inhibitor suppressed the formation of tetraploid cells (Fig. 4C). Furthermore, treatment with either inhibitor led to the reestablishment of equatorial RhoA localization (Fig. 4D, E). Together these data indicate that BRAF V600E acts through Rac1 to inhibit RhoA and cause failure of cytokinesis.
Supernumerary centrosomes are observed in BRAF V600Eexpressing cells. In assessing how Rac1 activity could be upregulated in BRAF V600E -expressing cells, we discovered that mitotic spindles in BRAF V600E -expressing cells showed structural organizations consistent with the presence of multiple, clustered centrosomes at the same spindle pole. Extra centrosomes increase microtubule nucleation, which in turn stimulates the activity of Rac1 66,67 . To determine whether extra centrosomes were present in BRAF V600E -expressing cells, we performed quantification of centrosomes via gamma-tubulin staining in S phase cells.
Here, we observed a significant increase in cells with more than 2 centrosomes in the BRAF V600E -induced population ( Supplementary Fig. 5A, B). Gamma-tubulin can stain fragments, derived from previously intact centrosomes, that can continue to nucleate microtubules 68 . To address this possible artifact and confirm the presence of supernumerary centrosomal components in BRAF V600E -expressing cells, we stained for the centriolar marker Centrin-2 in mitotic cells (Fig. 5A). Supernumerary Centrin-2-positive centrioles (>4 centrioles per cell) were observed in 22% of BRAF V600E -expressing cells, which is an eightfold increase as compared to control cells (Fig. 5B). In some cases, unpaired, single centrioles, suggestive of centriole overduplication were observed (Supplementary Fig. 5C). To determine if supernumerary centrioles were able to accumulate pericentriolar material (PCM) and nucleate microtubules, we stained for the PCM marker Pericentrin and alpha-tubulin to visualize microtubules. Cells that had three or more foci of centrioles were assessed so PCM at a supernumerary centriole could be distinguished. We found that 91% of cells had Pericentrin at three or more foci. (Supplementary Fig. 5D). For alpha-tubulin staining, cells that had two or more foci of centrioles at a spindle pole were assessed so microtubules at a supernumerary centriole could be distinguished. We found that 79% of cells had microtubules emanating from two or more foci ( Supplementary Fig. 5E). These data indicate that supernumerary centrioles arose as a consequence of BRAFV600E expression and many of these centrioles were competent to accumulate PCM and nucleate microtubules. Supernumerary centrioles were also present in BRAF V600Eexpressing human Mel-ST cultured melanocytes ( Supplementary  Fig. 6A, B) and BRAF V600E -expressing zebrafish melanocytes (Fig. 5C, D, Supplementary Fig. 6C, D), supporting the relevance of observations in RPE-1 cells to melanocyte and melanoma biology. To determine whether supernumerary centrioles arose due to BRAF V600E -dependent MAPK signaling activity, we treated BRAF V600E -expressing cells with the MEK inhibitor trametinib and ERK inhibitor SCH772984. Both inhibitors suppressed the increase in centrioles (Fig. 5A, B), indicating that BRAF V600Edependent overactivation of MAPK signaling led to supernumerary centrioles.
Supernumerary centrioles contribute to RhoA downregulation and BRAF V600E -induced WGD. Our findings indicated that BRAF V600E caused RhoA downregulation and failure of cytokinesis as well as an increase in centrioles. We sought to determine whether the increase in centrioles contributed to RhoA downregulation and tetraploid cell formation. First, we examined if BRAF V600E -expressing cells with extra centrioles had reduced RhoA localization as compared to BRAF V600E -expressing cells with normal centrioles. BRAF V600E -expressing cells with a normal number and arrangement of centrioles had reduced RhoA staining; however, BRAF V600E -expressing cells with extra centrioles had an even greater reduction in RhoA localization (Fig. 6A, B). To establish that the reduced localization of RhoA in these cells was dependent on extra centrioles, we treated cells with centrinone, a PLK4 inhibitor that blocks centrosomal duplication 69 . Centrinone treatment suppressed the formation of extra centrioles in BRAF V600E -expressing cells (Fig. 6C). The centrinone-treated BRAF V600E -expressing cells had higher RhoA equatorial localization as compared to control BRAF V600Eexpressing cells (Fig. 6D, E). This recovery of RhoA localization was evident not only when centrinone treatment was coincident with BRAF V600E expression, but also when centrinone treatment was limited to late G1 and early S. Centrinone treatment also partially suppressed the BRAF V600E -driven formation of tetraploid cells (Fig. 6F). Thus, suppression of supernumerary centrioles in BRAF V600E -expressing cells recovered not only RhoA localization but also promoted cytokinesis. Taken together, these data indicate that the reduction of RhoA localization and cytokinesis in BRAF V600E -expressing cells is partially dependent on the BRAF V600E -driven increase in centrioles. Because the suppression of supernumerary centrioles in BRAF V600E -expressing cells did not fully restore RhoA localization and cytokinesis, a separate, centriole-independent mechanism driven by BRAF V600E also reduces RhoA localization and cytokinesis.
p53 blocks cell cycle progression of BRAF V600E -induced tetraploid cells. Our finding that BRAF V600E can cause failure of cytokinesis and lead to tetraploidy suggests that this may underlie the genome doubling events that are evident in BRAF V600Emutated melanomas. However, for BRAF V600E -expressing cells to contribute to tumor formation, they would likely have to overcome the G1 phase arrest that tetraploid cells experience 21,70 . This tetraploid arrest can be triggered by Hippo pathway activation, which itself is activated by supernumerary centrosome-dependent activation of Rac1 46 . Hippo pathway activation, in turn, activates p53, which has been shown to mediate arrest of tetraploid cells 70,71 . Additionally, inactivation of p53 is strongly correlated with WGD in clinical samples, supporting the possibility of a p53-dependent arrest in tumors 16 .
To more directly address a p53-dependent arrest, we used Crispr/Cas9-mediated genome editing to knock out P53 in our BRAF V600E -inducible RPE1-FUCCI cell clone. We isolated a clone in which both P53 alleles were targeted and P53 protein expression abrogated ( Supplementary Fig. 7C, D). These cells had DNA content profiles similar to P53 wild-type parental cells ( Supplementary Fig. 7E, F). As compared to parental BRAF V600Einducible RPE1-FUCCI cells, BRAF V600E -induclible P53 −/− RPE-1 FUCCI cells had a lower fraction of G1 tetraploids following BRAF V600E expression ( Supplementary Fig. 7G, H), consistent with the notion that an arrest of G1 tetraploids was bypassed in P53 −/− cells. Such a failure to arrest would be evidenced by the presence of Geminin-GFP-positive S/G2/M cells with a >4N DNA content. Such cells were present at a much higher fraction in BRAF V600E -induclible P53 −/− RPE-1 FUCCI cultures and were observed after Dox withdrawal in extended cultures (Supplementary Fig. 7I, J), suggesting that, once generated, tetraploid cells could continue to cycle in a P53 −/− background. To more directly determine if a P53-dependent arrest prevents BRAF V600Eexpressing tetraploids from entering the cell cycle, we isolated G1 tetraploids after release from synchronization, cultured them for 24 h, then assessed cell cycle progression. As measured by Geminin-GFP expression, BRAF V600E -expressing RPE1-FUCCI cells showed little to no progression (Fig. 7C, D). By contrast, nearly 15% of BRAF V600E -expressing p53 −/− RPE-1 FUCCI cells were Geminin-GFP-positive. The majority of these Geminin- GFP-positive cells had DNA content reflective of progression into S/G2/M cell cycle phases (Fig. 7C). Together these data indicate that BRAF V600E -induced tetraploid cells arrest in G1, and this arrest is alleviated by loss of P53. Our analysis of human tumor samples confirmed that loss of p53 pathway activity was strongly correlated with WGD, including in tumors that harbor BRAF mutations and other mutations that activate RAS/MAPK signaling ( Supplementary Fig. 8).

Nascent tumor cells in BRAF V600E
-driven zebrafish melanomas are tetraploid and have higher ploidy. Our data indicate that BRAF V600E can induce tetraploidy in melanocytes, and these melanocytes are prevented from progressing further by a p53dependent block. Since BRAF V600E and loss of p53 cooperate to form melanomas, and because BRAF V600E causes tetraploidy as seen in our in vivo and in vitro models, we were interested in understanding whether BRAF V600E -generated tetraploid cells serve as intermediates in melanomagenesis in our zebrafish model, potentially as cells of origin. This would be consistent with findings in tumor types with frequent WGD, in which tumors that have undergone a WGD event are thought to have undergone that event early in tumor progression 3,73 . To determine if tetraploid cells are present in the earliest stages of tumor formation, we took advantage of the zebrafish model in which early melanomas can be identified because of their reactivation of the neural crest gene crestin 74 . Using a Tg(mitfa:BRAF V600E ); p53(lf); Tg(crestin:EGFP) strain that marks tumor initiating cells, we identified zebrafish with early tumors (<20 cells), dissected these tumors and performed flow cytometry to determine DNA content of crestin:EGFP-positive tumor cells. The crestin:EGFP-positive cells were mostly tetraploid, and several octoploid cells were also observed (Fig. 7E). Notably, none of the cells were diploid, indicating that cells were tetraploid very early in tumor formation and it is likely that the cell of origin was tetraploid. Furthermore, the octoploid nascent tumor cells were mostly mononucleate (Fig. 7E) and thus different from the octoploid, binucleate melanocytes present in Tg(mitfa:BRAF V600E ); p53(lf) strains. We speculate that these mononuclear, octoploid tumor cells arose from tetraploid cells of origin, and were cycling tetraploid cells in G2 and M phases. These data suggest that WGD can be present at the time of tumor initiation and potentially support tumor progression.

Discussion
Our results indicate that BRAF V600E , and RAS/MAPK signaling in general, can cause the WGD that is a hallmark of melanomas and other tumor types. BRAF V600E causes WGD by suppressing the activity of RhoA, leading to failure of cytokinesis. This suppression stems, in part, from supernumerary centrosomes that are formed as a result of BRAF V600E activity. Supernumerary centrosomes are known to activate Rac1, and we found that Rac1 activity was necessary for BRAF V600E -dependent WGD. Taken together, our data support a model in which BRAF V600E causes the formation of supernumerary centrioles and centrosomes, leading to the activation of Rac1, which in turn causes inhibition of RhoA and failure of cytokinesis. The binucleate, tetraploid cells that result arrest in G0/G1 unless cells have an underlying mutation, in our models it is loss of P53, that abrogates the arrest and enables further cell cycle progression. Thus, in addition to stimulating cell cycle progression, suppressing cell death and providing other tumor-promoting activities, BRAF V600E can cause WGD, which has been shown to support tumorigenesis and tumor progression.
A key component of this model is the formation of supernumerary centrosomes in BRAF V600E -expressing cells. The requirement for BRAF V600E activity in G1/S coincides with the timing of centrosomal duplication in most cell types. Furthermore, gamma-tubulin staining in BRAF V600E -expressing cells showed an increase in centrosome numbers in cells that had only progressed into S and G2 phases after BRAF V600E induction in G1. Together these observations suggest that the defect caused by BRAF V600E is that of centrosomal overduplication. A similar phenotype has been observed upon BRAF V600E overexpression in established melanoma cell lines, although such cells had a high background of underlying centrosomal abnormalities 75,76 . More recently, direct staining of melanoma samples suggests that supernumerary centrosomes in patient samples arise predominantly through overduplication 77 . The mechanism by which BRAF V600E could cause overduplication is not clear, although the ability of centrinone to suppress BRAF V600E -induced supernumerary centrioles indicates that this mechanism is PLK4dependent. Our data also indicate that supernumerary centrioles accumulate pericentriolar material (PCM) and have microtubule organizing center (MTOC) activity once they are formed. This is surprising because, under normal circumstances, newly-formed centrioles need to traverse through mitosis to accumulate PCM and nucleate microtubules 78 . How precocious maturation of BRAF V600E -induced supernumerary centrioles could occur is not entirely clear, although a combination of studies suggest premature centriole disengagement might be important. RAS/MAPK pathway activity, such as that induced by BRAFV600E, is a wellestablished cause of replication stress 79,80 . Recently, it has been shown that replication stress causes premature centriole disengagement, leading to the formation of multipolar spindles that show microtubule nucleation on prematurely disengaged centrioles 81 . Premature centriole disengagement caused by disruption of Cep57 and PCM components also leads to precocious maturation, suggesting that premature disengagement can generally enable precocious maturation and MTOC activity 82 . Thus, taken together, RAS/MAPK-induced replication stress caused by BRAF V600E expression could lead to premature centriole disengagement followed by precocious maturation and MTOC activity. As noted, the effect of supernumerary centrosomes only accounts for some of the RhoA reduction and cytokinesis failure upon BRAF V600E induction. Nonetheless, our findings together with the association of BRAF V600E with centrosomal amplification in  papillary thyroid, colorectal and other cancers suggests there may be a broad link between BRAF V600E -induced supernumerary centrosomes and WGD 83,84 .
Our findings begin to address the fate of cells that undergo WGD through cytokinesis failure. Zebrafish melanocytes and RPE-1 cells that have undergone cytokinesis failure arrest in G1 as binucleate tetraploid cells. However, in established zebrafish melanomas and late-stage human tumors that have previously experienced a WGD event, tumor cells go through the cell cycle, are predominantly mononuclear and do not indefinitely undergo cytokinesis failure. This raises the questions of how cells having just undergone a WGD event overcome the G1 block and, once they do, divide productively without creating giant, multinucleated cells. Newly-generated G1 binucleate tetraploid cells undergo a P53-dependent arrest, as evidenced by the progression into S phase of P53-mutant RPE-1 G1 tetraploids. The polyploid nature of Tg(mitfa:BRAF V600E ); p53(lf) melanocytes indicates that this arrest also occurs in the zebrafish model. However, these zebrafish melanocytes are found as binucleates with most nuclei appearing to have gone through a single S phase without any further mitosis. This suggests that additional blocks may exist to prevent such cells from progressing further into the cell cycle, and such blocks would have to be overcome during tumorigenesis. If such cells were to progress and continue cycling, then conditions would have to exist to prevent them from stalling tumor growth as multinucleated cells. One possible condition is that the penetrance of BRAF V600E -induced cytokinesis failure is incomplete as in RPE-1 cells. Enough cytokinesis failure to enable WGD would be present, but not enough to prevent the amount of cell divisions required for tumor growth. Another possible condition is the attenuation of cytokinesis failure following a WGD event. RhoA activity has been shown to be upregulated in tumors [85][86][87] , and this could be an adaptation that prevents pervasive cytokinesis failure that would be detrimental to tumor progression.
Our finding that nascent zebrafish melanoma cells were tetraploid or had higher ploidy supports the notion that BRAF V600E -induced WGD can be present at the time of tumor initiation. In cancer types that have a high prevalence of WGD, such as melanoma, WGD in tumors frequently occurred early in tumor formation and might have been present at the time of tumor initiation and clonal outgrowth 3,73 . This raises the question of whether BRAF V600E , which is present in~80% of benign nevi 88,89 , can cause WGD at the earliest stages of tumor formation and possibly in benign lesions. Although cells in common cutaneous nevi are primarily diploid 90,91 , several observations suggest that cytokinesis failure and associated WGD can occur in these cells. Cultures of nevocytes have been shown to contain binucleated cells 92 . Furthermore, the presence of binucleate and multinucleated giant cells in nevi is not uncommon 93 , and polyploid cells are observed at a low fraction in many nevus samples 94 . Together these observations suggest that some level of cytokinesis failure occurs in nevi and may be caused by BRAF V600E . The prevalence of early WGD in melanomas as compared to its relative absence in nevi, at least its absence when nevi begin clonal outgrowth, suggests there is some advantage to WGD in tumor formation. The ability to sample various protumorigenic genomic configurations may underlie the benefit of WGD 95 as would the ability of WGD to make haploid regions of a nascent tumor cell diploid and thus protect these regions from mutations incurred in essential genes 24 . Lastly, although the focus has been on WGD supporting tumor initiation, it is clear that many tumors analyzed bioinformatically, including the majority (~60%) of melanomas, show no evidence of having undergone WGD. However, this does not mean WGD is irrelevant in such tumors. Recent evidence suggests that WGD in melanomas can be late truncal and even occur privately in metastases 96,97 . Therefore, while WGD may not be necessary in all tumors for initiation, it nonetheless could play a role in disease progression. It is worth noting that the WGD observed in nevocytes, primary tumors and metastases overlaps with the presence of BRAF V600E or other RAS/MAPK-activating mutations that could support WGD in such lesions.

Methods
Zebrafish strains and husbandry. Zebrafish were handled in accordance with protocols approved by the University of Massachusetts Medical School IACUC (A-2171). Strains were maintained at 28°C as described by Westerfield 98 . AB was used as the wild-type strain. The following mutations were used: p53(zdf1) 99 , mitfa(w2) 100 , alb(b4) 101 and are designated throughout as "lf" for loss-of-function mutation. The integrated transgene Tg(mitfa:BRAF V600E ) expresses oncogenic human BRAF V600E under the control of the zebrafish pigment cell-specific mitfa promoter 38 . Equal numbers of male and female animals were used in experiments.
Cell line generation. RPE-1 FUCCI cells were grown in DMEM: F12 media containing 10% tetracycline-free FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Mel-ST cells were grown in DMEM media containing 5% tetracyclinefree FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained at 37°C with 5% CO 2 atmosphere. TetR was expressed in cells using pLenti CMV TetR Blast (Addgene #17492). Transduced cells were selected with 5 µg/ml blasticidin and bulk cells expressing the TetR protein were clonally isolated in 96-well plates using FACS (BD-Aria). BRAF V600E and BRAF WT were expressed in cells using Gateway-compatible lentiviral constructs (pLenti CMV/TO puro DEST, pLenti CMV/TO neo DEST; gifts from Eric Campeau and Paul Kaufman 102 ). Lentivirus carrying BRAF WT or BRAF V600E construct was generated by transfection of HEK-293T cells, with appropriate packaging plasmids (pMD2.G and psPAX2) using lipofectamine 2000, according to the manufacturer's instructions. RPE-1 and Mel-ST cells were infected for 48 h with virus carrying a BRAF construct in the presence of 8 µg/ml polybrene, washed, and allowed to recover for 24 h before selection. Cells were selected with the appropriate antibiotic selection marker (5 µg/ml puromycin or 1 mg/ml neomycin) and clonally isolated in 96-well plates using FACS (BD-Aria). Doxycycline was used at 1 µg/ml to induce the expression of BRAF.
Synchronization and ploidy analysis. To turn on BRAF V600E in a synchronized cell population, we serum starved RPE-1 FUCCI cells with 0.1% serum for 48 h. Serum-starved cells were released into media with 10% serum until they reached the next G1 (~33 h). BRAF V600E was turned on using doxycycline, and cells were resynchronized at G1/S using thymidine (2.5 mM). Following thymidine washout cells were allowed to progress through the cell cycle, and ploidy was analyzed in next G1 (16 h after thymidine release) using Hoechst incorporation (2.5 µg/ml) in Live cell imaging. For live cell imaging, RPE-1 H2B-GFP-transfected cells were grown on glass-bottom 12-well tissue culture dishes (MatTek) and imaged on a Nikon TE2000E inverted microscope equipped with a cooled Hamamatsu Orca-ER CCD 11 camera and the Nikon Perfect Focus system. The microscope was enclosed within an incubation chamber that maintained an atmosphere of 37°C and 3-5% humidified CO 2 . GFP and phase-contrast images were captured at multiple locations every 5 min with either 20× or 40× Nikon Plan Fluor objectives. All captured images were analyzed using NIS-Elements software. For imaging, cells were synchronized with thymidine and imaged for 48 h after release. Each cell going through mitosis was observed and cytokinesis was scored by phase contrast imaging. To verify cytokinesis failure and to confirm that the nuclei stayed within one cell, cells were carefully traced for at least ten frames. To quantify mitotic duration, each frame beginning from the start of nuclear envelope breakdown to anaphase onset was counted.
Immunofluorescence and imaging. RPE-1 BRAF V600E cells were grown directly on collagen IV (Sigma) -coated coverslips, fixed in 3.7% formalin, permeabilized using 0.1% Triton X-100, and treated with 0.1% SDS. They were blocked in 1% BSA and then incubated with primary antibody diluted in blocking solution in a humidity chamber at 4°C overnight, washed with 1X PBS, and incubated with secondary antibody. Cells were mounted using mounting media containing DAPI (Vector Laboratories). All secondary antibodies were Alexa Fluor conjugates (488, 555, and 647) (Thermofisher) used at a 1:500 dilution. All images were acquired on a Leica DM 600 inverted microscope at 100× magnification and analyzed using Leica LAS X software (v. 3.7.1.21655). Z stack images were taken at 0.20 µM each. For melanocyte binucleate counts and immunofluorescence staining, zebrafish adult dorsal scales were fixed using 4% paraformaldehyde for 2 h, washed with PBST (PBS + 0.1% Triton X) and water then blocked in 1%BSA/PBS for 30 min prior to primary antibody incubation. Affinity-purified anti-Mitfa antibodies were used at a 1:100 dilution for staining. All images were acquired on a Nikon Eclipse Ti A1R-A1 confocal microscope and analyzed using NIS-Elements software.
Cyclin D1 staining for flow cytometry. RPE-1 FUCCI cells were trypsinized, washed with 1XPBS and fixed in 2% PFA at RT for 15 min. Cells were then washed with PBS/1%FBS and permeabilized with 100% methanol (for CYCLIN D1). Cells were washed with PBS, incubated with Alexa Fluor 647-conjugated primary antibodies for 60 min at room temperature, washed and stained with 1:1000 DAPI for 15 min before flow cytometry (BD-LSR).
CRISPR cell line generation. Oligos targeting p53 were annealed and inserted into lentiCRISPR v2 (gift of Feng Zhang) as described previously 103,104 The lentiviral packaging plasmids pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) were used for transfection using lipofectamine. For lentiviral transduction, cells (300,000) were plated in a 6 well plate the day before transduction. Lentivirus was harvested and added to OptiMEM supplemented with 8 µg/ mL polybrene. Media was changed 24 h after transduction to remove polybrene. Media supplemented with 5 µg/mL puromycin (Sigma Aldrich) or was changed 48 h after transduction to select lentiCRISPRv2 transduced cells. Following transduction with p53-targeting lentiCRISPRv2 containing lentivirus, antibioticresistant cells were selected then clonally isolated by FACS in 96-well plates. Clones were then assayed for indels via the surveyor assay (706020, IDT). Cells that showed indels were then cloned into the pGEM-T Easy vector (Promega), and colonies were picked using blue/white screening then sequenced. The p53 gRNA sequence was CCCCGGACGATATTGAACAA. Primers used for sequencing indels were as follows: Forward -5′ GTAAGGACAAGGGTTGGGCT 3′ Reverse -5′ GAAGTCTCATGGAAGCCAGC 3′ Flow cytometry of zebrafish melanocytes, normal tissue and tumors. To isolate zebrafish melanocytes for flow cytometry, 4-to 6-month-old fish were treated for 5 min with the anesthetic tricaine methanesulfonate. Scales were plucked from the dorsal anterior region of fish and put in 0.9× PBS. Cells were dissociated using TH liberase (Roche) for 30 min by constant agitation at 37°C. Cells were immediately fixed for 2 h in 4% paraformaldehyde. Following fixation, cells were washed and permeabilized 3× with 0.1% Triton X/PBS and immediately stained with DAPI (1:1000). Cells were then filtered through a 40 µM mesh filter and spun down at 2000rpm. Analysis for GFP+ and DAPI+ cells was performed on the Amnis Flowsight using IDEAS software (v. 6.0). For ploidy analysis of zebrafish tumors, melanomas from Tg(mitfa:EGFP); (mitfa:BRAF V600E ); p53(lf);alb animals were removed, homogenized in PBS, and stained and analyzed in the same manner as described above. For normal tissue analysis, the caudal peduncle and fin were dissected and homogenized.
Confocal densitometry. Zebrafish scales were obtained and stained with anti-Mitfa antibody and DAPI as described above. Melanin pigment interferes with quantitative UV-based imaging so scales were bleached prior to staining. Mitfapositive melanocyte nuclei were identified, and Z stacks of the DAPI signal of these nuclei were obtained. The same distance between Z slices (0.37 μM) and pixel intensity lookup table were used for each nucleus measured. In analyzing each slice, nuclear boundaries were specified, and pixel intensity values within the nuclear area measured. Pixel intensities for all slices of one nucleus were summed to derive a raw DNA content. Ploidy was estimated using nearby Mitfa-negative nuclei as 2 N controls. Nuclei in the same binucleate cell are typically arranged as mirrorimage pairs, and this orientation allowed us to identify nuclei of binucleate cells in the absence of melanin pigment.
Melanocyte density assay. Four-to six-month-old fish were treated for 5 min with the anesthetic tricaine methanesulfonate and epinephrine, which contracts melanosomes to the central cell body of melanocytes (Goodrich and Nichols, 1931), thereby resolving overlapping cells. Scales were plucked from the dorsal anterior region of fish from the scale rows adjacent to the dorsal midline row. Scales were immediately fixed for ≥30 min in 4% paraformaldehyde. After fixation, scales were flat-mounted and melanocytes counted. Area was estimated by multiplying maximal antero-posterior and left-right distances of the scale.
Data collection and statistical analysis. Microsoft Excel (v. 16.16.27) and Graphpad Prism (v. 8.0) were used for data collection. Significance calculations were performed on samples collected in a minimum of biological triplicate. P values from two-tailed Student's t tests or ANOVA were calculated for all comparisons of continuous variables. All further significance tests were performed in GraphPad Prism (v. 8.0). A P value < 0.05 was considered significant.
Drug treatments. Cells were synchronized as described with serum starvation and thymidine arrest. Treatment of cells with small molecule compounds was performed as follows: G1/S/G2/M-drug was added 33 h following serum addition and coincident with doxycycline; S/G2/M-drug was added following thymidine washout; G1/S-drug was added 39 h following serum addition and washed off 12 h later; G1-drug was added 33 h following serum addition and washed off 6 h later; early G1drug was added 33 h following serum addition and washed off 2 h later. Small molecule inhibitors used were-Vemurafenib, 1 µM (S1267, Selleck RhoA and Anillin intensity measurements. Fluorescence intensities were quantified using ImageJ as previously described 105 . Briefly, Z-stack images were loaded on ImageJ (v. 1.53a) and the sum intensity projection was obtained using the Z-stack function. RhoA and Anillin equatorial fluorescence intensities were obtained by measuring the intensity profile of the fluorescence signal along a line manually placed along the cell equator, parallel to the anaphase DNA position. The mean fluorescence intensity was obtained by averaging the two intensity values at each side of the furrow. The mean background signal was obtained by averaging the signal of three manually selected circular regions with a diameter of 50 pixels outside of the cell and the value was subtracted from the equatorial intensities.
Growth and apoptosis assays. To perform growth curves, 20,000 cells were plated in 12-well plates and appropriate samples were treated with inhibitors and doxycycline. Cells were counted every 24 h over 4 days using the hemocytometer. Cell death following inhibitor treatment was assessed by the caspase glow assay according to the manufacturer's instructions (G8090, Promega).
Whole-genome doubling analysis. Whole-genome doubling (WGD) data for each cohort were obtained for TCGA samples 106 and MSK-IMPACT samples 16 , and mutation data were obtained from cBioPortal 107,108 . Associations of WGD with BRAF mutations as well as ANY RAS/MAPK pathway genes (BRAF, NRAS, KRAS, HRAS, NF1, MAP2K1, MAP3K13 and PTPN11) were analyzed. Associations of WGD with BRAF or ANY RAS/MAPK mutations together with TP53 or CDKN2A mutations and copy number losses were also analyzed, with the rationale that our data support a combination of RAS/MAPK activation plus TP53 pathway inactivation enable progression of cells that undergo WGD.